Therapeutic proteins which are generally administered by intravenous injection may be immunogenic, relatively water insoluble, and may have a short in vivo half-life. The pharmacokinetics of the particular protein will govern both the efficacy and duration of effect of the drug. It has become of major importance to reduce the rate of clearance of the protein so that prolonged action can be achieved. This may be accomplished by avoiding or inhibiting glomerular filtration which can be effected both by the charge on the protein and its molecular size (Brenner et al., (1978) Am. J. Physiol., 234, F455). By increasing the molecular volume and by masking potential epitope sites, modification of a therapeutic polypeptide with a polymer such as polyethylene glycol (PEG) has been shown to be efficacious in reducing both the rate of clearance as well as the antigenicity of the protein. Reduced proteolysis, increased water solubility, reduced renal clearance, and steric hindrance to receptor-mediated clearance are a number of mechanisms by which the attachment of a PEG polymer to the backbone of a polypeptide may prove beneficial in enhancing the pharmacokinetic properties of the drug. Thus Davis et al., U.S. Pat. No. 4,129,337 discloses conjugating PEG to proteins such as enzymes and insulin to produce a less immunogenic product while retaining a substantial proportion of the biological activity.
PEG modification requires activation of the PEG polymer that is accomplished by the introduction of an electrophilic center. The PEG reagent is now susceptible to nucleophilic attack, predominantly by the nucleophilic epsilon-amino group of a lysyl residue. Because of the number of surface lysines present in most proteins, the PEGylation process can result in random attachments leading to mixtures which are difficult to purify and which may not be desirable for pharmaceutical use.
There are a large variety of PEG reagents that have been developed for the modification of proteins. This involves the covalent attachment of a PEG molecule via the formation of a linking group between the PEG polymer and the protein (see for example Zalipsky, et al., and Harris et. al., in: Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21 and 22). Some of these reagents are, to various degrees, unstable in the aqueous medium in which the PEGylation reaction occurs. In addition, the conjugation process often results in the loss of in vitro biological activity that is due to several factors foremost of which being a steric interaction with the proteins active sites. A desired property therefore of a new reagent would be one that is not susceptible to degradation in an aqueous medium and one that may be employed to affect the site specific modification of a protein. A PEG aldehyde may be considered such a reagent. For site specific N-terminal pegylation see Pepinsky et al., (2001) JPET, 297, 1059 (Interferon-□-1a) and U.S. Pat. No. 5,824,784 (1998) to Kinstler et al., (G-CSF). The use of a PEG-aldehyde for the reductive amination of a protein utilizing other available nucleophilic amino groups, is described in U.S. Pat. No. 4,002,531 (1977) to Royer, in EP O 154 316, by Wieder et al., (1979) J. Biol. Chem. 254, 12579, and Chamow et al., (1994) Bioconjugate Chem. 5, 133.